Insect cells infected with tsB1074, a temperature-sensitive mutant of Autographa californica nuclear polyhedrosis virus, exhibit a "single-cell-infection" phenotype whereby the infection progresses through the very late phase culminating in occlusion body formation, but neighboring cells do not become infected. Marker rescue mapping and DNA sequencing correlated a single nucleotide substitution within the baculovirus gp41 gene with the temperature-sensitive phenotype of tsB1074. The product of the gp41 gene, GP41, is an O-glycosylated protein found in occluded but not budded virions [M. Whitford and P. Faulkner (1992) J. Virol. 66, 3324-3329]. However, budded virus was not produced in tsB1074-infected cells at the nonpermissive temperature of 33 degrees, indicating an additional role for GP41 in budded virus formation. Electron microscopy revealed that nucleocapsids were produced but retained in the nucleus of tsB1074-infected cells at 33 degrees. Thus, GP41 was required for the egress of nucleocapsids from the nucleus in the pathway of budded virus synthesis.